FR2775001A1 - NUCLEIC ACID COMPRISING THE SEQUENCE OF A PROMOTER INDUCIBLE BY STRESS AND A SEQUENCE OF A GENE ENCODING STILBENE SYNTHASE, CELL AND PLANT TRANSFORMED THEREWITH NUCLEIC ACID - Google Patents

Abstract

The invention concerns plants with improved resistance to certain stilbene-sensitive pathogenic agents, and more particularly a set of constructs combining a plant promoter inductible by biotic stress, generated in particular by said pathogens, and gene(s) coding for a stilbene synthase.

Description

 The present invention relates to plants having improved resistance to certain pathogens sensitive to stilbenes, and more particularly relates to a set of constructions associating a plant promoter inducible by a biotic stress, generated in particular by said pathogens, to one or more genes (s). ) coding for a stilbene synthase.

 Much of the world crop crop is constantly being destroyed by pests and pathogens. Among the possibilities to reduce or prevent the attack of plants cultivated by these parasites, chemical control (phytosanitary treatments) is the most used method.

However, the application of chemicals is not without consequences for the environment and sometimes poses technological problems such as the appearance of new strains resistant pathogens or, in the field of oenology, the difficulties that may arise during fermentations (the use of inhibitors of sterol biosynthesis may block the growth of yeasts at the end of fermentation) or the presence of chemicals sometimes found in wines such as procymidone, anti-Botrytis product.

To overcome the disadvantages of chemical control, the control method of improving the resistance of cultivated plants to diseases caused by these pathogens was considered. It is possible, for example, in a first approach, to obtain this improvement by sexual route, by conventional genetics, by hybridizing the plants whose resistance is to be improved with tolerant varieties. Nevertheless, this approach is not always feasible (natural variety tolerant not known) or is not authorized by the legislation as for example in viticulture by the French legislation on the Appellations of origin
Controlled (AOC) which limits the grape varieties to be used for a given appellation.

 In a second approach, it is possible, using modern techniques of cellular and molecular biology, to integrate into the genome of the plant one or more homologous or heterologous genes allowing the overexpression or the expression of a molecule of interest of a protein nature in order to increase the production of a metabolite or a metabolic pathway or to open a new biosynthetic pathway or to synthesize a new molecule making it possible for example to increase the opening of a new pathway of biosynthesis, allowing for example to increase the resistance of the plant by strengthening its defense mechanisms vis-à-vis the pathogens involved.

In plants, these defense mechanisms are of several kinds. Some may be considered passive and are related to the physico-chemical characteristics of the cells, epidermal tissues and / or organs of the plant. The others belong to the dynamics of gene-gene interactions (plant resistance and pathogen avirulence genes, mechanisms of host-pathogen interactions). These interactions can lead to the development of the hypersensitivity reaction (rapid death of plant cells around the point of infection to block the colonization of the plant by the microorganism) but also to the synthesis and accumulation of a whole series of compounds. Of these, some may be parietal constituents, involved in the formation of a physical barrier around the point of infection (callose, lignin, hydroxyproline-rich protein: HRGP, etc.), other compounds may be molecules with more or less well-defined antimicrobial functions (phytoalexins, proteins associated with pathogens: PR proteins (Pathogenesis Related Proteins), etc.). Among the phytoalexin-like molecules that are synthesized and accumulated by plants during, for example, host-pathogen interactions, stilbenes that are toxic, especially for microorganisms, are found in particular. The term stilbene refers to a group of chemicals having the trans-diphenyl-1,2-ethylene backbone as a common basic structure, with resveratrol and pinosilin being among the simplest. This basic skeleton is synthesized in plants by a stilbene synthase or related enzymes, from substrates such as malonyl-CoA, cinnamoyl-CoA or coumaroyl-CoA, substances found in all plants (precursors flavonoids). Stilbene synthase genes or related enzymes have been isolated, sequenced and cloned, including from peanuts, orchids and grapes. From these genes it was possible to carry out the transformation of plants such as potato, alfalfa or tobacco, having, in comparison with the non-transformed plants, a higher resistance to attack by pathogens (EP-309862; EP-648839; MELCHIOR, F. et al.,
Arch. Biochem. Biophys. 1991, 288, 2, 552-557; Wiese, W. et al., Plant Mol. Biol. 1994, 26, 2, 667-677; Hain, R. et al., Nature 1993, 361, 153-156).

 The expression or the overexpression of these molecules with antimicrobial functions can make it possible to obtain a natural resistance of the plants in response to stress, in particular microbial stress. However, a constitutive overexpression of this type of protein can not be envisaged without inconvenience for the plant (energy cost, slowing down of growth, etc.) (FISCHER, R. et al., The plant Journal 1997, 11, 3, 489498 ).

 On the other hand, in the case of certain plants such as grapevine or herbaceous plants, stilbenes are found only in certain healthy tissues and at very low concentrations. On the other hand, following an infection or injury, these stilbenes increase strongly at the infected or injured site, the stilbene synthase genes being inducible under biotic or abiotic stress conditions (eg wounds, ultraviolet, etc. .).

 Nevertheless, this regulation is rarely present in plants of agronomic interest or when it is present, it may not be sufficiently effective. For example, in the case of grapevine, the study of phytoalexin synthesis has shown the presence of stilbenes, including resveratrol, in healthy tissue only for wood.

In the tissues of the grape berry, the presence of stilbenes is found when, on the one hand, the berry is under stress like attack by a pathogen (Botrytis cinerea for gray rot or Plasinopora viticola for Mildew) on the other hand, only until the veraison of the young fruit. On the other hand, the concentration of stilbenes strongly decreases from veraison to maturation. However, the damage due for example to Botrytis are rarely encountered until veraison but rather when one is near the maturation of the bay. Therefore it is necessary to control the expression of stilbene synthase genes by strong promoters, escaping the natural regulation of the gene, promoters to be inducible and in particular inducible by the stress itself. This is precisely the object of the present invention.

 The present invention relates to nucleic acids comprising the promoter sequence of an alfalfa PR protein associated with at least one sequence of a gene encoding a stilbene synthase.

 The invention particularly relates to nucleic acids according to the invention, characterized in that the promoter of an alfalfa PR protein is an inducible promoter in plants, specific tissues or not, by a biotic or abiotic stress.

The invention also relates to nucleic acids according to the invention, characterized in that the promoter sequence of a PR protein of alfalfa is selected from the group comprising
a) the IND S1 sequence,
b) any sequence corresponding to a fragment of the IND S1 sequence and having a promoter sequence effect in plants.

 Alfalfa PR protein promoter sequences which have at least 80 k homology with the IND S1 sequence are preferred. Particularly preferred are those having at least 90 k or 95 W of homology with said sequence.

 The PR protein promoter sequences in alfalfa according to the invention were obtained from protein regulatory sequences of PR proteins by taking advantage of the incompatible response (HR hypersensitivity reaction) obtained in the host-parasite relationship between the alfalfa (Medicago sativa) and Pseudomonas syringae pv pisi to isolate regulatory sequences of genes responsible for this reaction.

 When alfalfa is attacked by Pseudomonas, the appearance of a reaction of the plant is observed in the area close to the necrosis caused by the bacterial infection.

 The plant material was therefore taken after the bacterial attack to form a cDNA library from the messenger RNAs produced in the neighboring areas of the necrosis. Polymerase chain reaction (PCR) amplification, using synthetic polynucleotides corresponding to conserved motifs of PR protein legume genes, yielded a radioactive probe which was then used to select transcripts in the library. cDNA. Of these, one of them (cDNA-PR7) was retained because, after sequencing, it had good homology with equivalent genes encoding PR proteins, known for other plants (see Figures 1 and ibis representing the general scheme of the promoter isolation method).

The analysis showed that it corresponded to a gene encoding a PR class 10 protein according to the VAN LOON classification (1994). That is why this gene has been called Ms PR10-1 (Medicago sativa PR protein class 10, clone 1). The isolated and cloned PR7 cDNA gave two probes thanks to the presence of an internal Bam HI site (B in FIG. 1). These, EB and BE (E corresponding to the Eco RI site in FIG. 1) designated respectively 5 'and 3', were used to screen a genomic alfalfa library. Among the clones obtained, recognized by the 5 'and 3' probes, one of them, C15, was selected and sequenced (6.1 kb). it also has a Bam HI site which makes it possible to obtain two new EB fragments of 2.4 kb and BE of 3.7 kb named respectively EB (g) and BE (g), g indicating the nature genomic fragments obtained (see Figure lbis). The EB (g) fragment, located 5 'of clone C15, comprising the promoter and a part of its Ms gene coding sequence
PR10-1 was inserted into the Eco RI and Healthy HI sites of the bluescript plasmid. The plasmid was linearized by means of a Pst I site located upstream of Bam HI in fragment E
B (g). On this fragment, a deletion of 3 'in 5' was carried out until obtaining the IND promoter sequence S1 (Figure 3). Free end ligation allowed repositioning another Bam HI site, internal to clone C15, at the end of the promoter sequence of the Ms PR10-1 gene. Under these conditions, 13 nucleotides of the coding sequence of the Ms PR10-1 gene, located upstream of this internal Bam HI site of the C15 clone, were thus integrated into the IND S1 promoter sequence, the whole can be isolated by digestion. Eco RI / Bam HI (see Example 1).

In the description, also means by PMs
PR10-1 any nucleic acid fragment of the IND S1 sequence with a promoter effect in plants and the S1 IND sequence with 13 nucleotides of the coding sequence of the Ms gene
PR10-1 as described below.

 The invention also relates to nucleic acids according to the invention, characterized in that the sequence of a gene coding for a stilbene synthase, whether homologous or heterologous, is chosen from genes isolated from the genomes of the peanut, orchid, vine and pine (EP-309 862, EP-464 461).

Among said nucleic acids, the nucleic acids encoding a grape stilbene synthase are preferred, in particular those described in the article by Hain, R. et al., Nature 1993, 361, 153-156 and in that of WIESE,
W. et al., Plant Mol. Biol., 1994, 26, 2, 667-677, the nucleic acid corresponding to the vstl sequence of said articles is most preferred.

 The nucleic acids allowing the expression of the gene (s) of stilbene synthase may of course comprise, in addition to said gene (s), sequences including polyadenylation at the 3 'end of the coding strand, as well as sequences enhancer of said gene or a different gene.

 Of course, the sequences of the nucleic acids will have to be adapted to ensure that the gene will actually be read in correct reading phase with the promoter and it will obviously be possible to plan to use, if necessary, several promoters of the same type as well as several enhancer sequences.

 It is also possible to express using the nucleic acids according to the present invention several stilbene synthase genes, either cascaded or carried by different expression systems.

 The nucleic acids according to the invention can be used for the production of expression systems in plants, which systems can be inducible and / or constitutive according to the tissues or organs of the transformed plant (see Examples 2, 3 and 4).

 The subject of the present invention is therefore also systems for expressing at least one stilbene synthase gene in plants, characterized in that they comprise at least one nucleic acid according to the invention.

Among the systems according to the invention, the transformation vectors and in particular the plasmid-type transformation vectors are preferred. Advantageously, said transformation vectors are characterized in that they can be transferred into strains of Agrobacterium.

The stilbene synthase genes that can be expressed by the nucleic acids according to the present invention are placed under the control of the PM promoter
PR10-1 in order to trigger in plants resistance mechanisms to pathogens that are sensitive to stilbenes, including resveratrol, pinosylvin or their glycosylated derivatives such as piceoid or oligomers such as viniferines. Amongst these stilbene-sensitive parasites, mention may be made of Botrytis cinerea,
Wine Plasmopora, Eutypa la, etc.

 Among the expression systems according to the invention, those characterized in that they are inducible in plants by biotic or abiotic stress are preferred.

 Among said biotic stresses according to the invention, biotic stresses generated by the attack of a stilbens-sensitive parasite such as a virus, a bacterium, a yeast or a fungus, in particular Botrytis cinerea or Plasmopora viticole, are particularly preferred.

 Among said abiotic stresses according to the invention, abiotic stresses generated by a mechanical wound, such as that caused in particular by an insect or by a physical phenomenon such as wind or gel, are particularly preferred.

 The subject of the present invention is also plant cells transformed by a system or a vector according to the invention. Advantageously, said plant cells are vine cells.

 The present invention also relates to methods for transforming plant cells using a microbiological process including the systems or vectors of the present invention.

 The invention furthermore relates to methods for obtaining plants expressing one or more stilbene synthase genes, characterized in that plant cells of said plants are transformed by means of a system or of a vector according to the invention, cells expressing said gene (s) are selected and a plant is regenerated from these cells.

 Among the most commonly used transformation methods, it is necessary to mention in particular the methods using Agrobacterium, whether it be Agrobacterium tumefaciens or Agrobacterium rhizogenes, biolistics or any other technique (electroporation, etc.).

These methods are known (REAM, W., 1989
NEGRETIU, I. and GHARTI-CHHETRI, GB, 1991; PUZZLE DELBART,
F., 1996; STANFORD, JC, 1990) and will not be described again in detail.

The technology, especially from plasmid systems, makes it possible to perform a first transformation of a strain of competent bacteria, in general
E. ccli, which makes it possible to clone and control the plasmid structure. The strain is then used to transfer the recombinant plasmids into agrobacteria strains which will then be used to transform the plant cells.

 Plants comprising an expression system or cells according to the invention form part of the invention.

 The plants obtained by carrying out the processes according to the invention also form part of the invention.

 Finally, the invention relates to the plants according to the invention, characterized in that they are plants of agronomic interest, including vines.

 Other features and advantages of the constructions and methods according to the present invention can be demonstrated in the following examples.

Legends of Figures
FIGS. 1 and 1a. General scheme showing the different steps of the method of isolating the inducible PMs PR10-1 promoter corresponding to the IND S1 sequence.

Figure 2: Presentation of the various clones isolated and corresponding to the Southern blot, hybridized with the 5 'and 3' portions of the cDNA-PR7, delimited by an internal Bam HI (B) site, detected in this cDNA.

 Restriction sites: E = Eco RI, B = Bam HI.

 The values shown in the figure are expressed in kb (kilo bases).

Figure 3: DNA sequence corresponding to the IND sequence
S1, genomic sequence isolated from the inducible alfalfa promoter PMs .PR10-1.

Figure 4: DNA sequence corresponding to the sequence of a vine stilbene synthase gene modified by the addition of an adapter (modified vstl).

 The modified part is represented in italics.

 The coding and non-coding parts (intron) are shown in upper and lower case letters, respectively.

FIG. 5: DNA sequence comprising the inducible PMs PR10-1 promoter sequence (corresponding to the IND S1 sequence in lower case) associated with the sequence of a vine stilbene synthase gene modified by the addition of an adapter ( modified vstl, corresponding to Figure 4). Between the two (box in the sequence) are (written in lower case, end of the promoter and in capital letters, beginning of the gene containing the translation initiation codon) the 13 nucleotides coming from an internal sequence of the coding phase of the Ms PR10-1 gene, ATG having been put in reading phase with the modified vstl gene, these nucleotides are therefore integrated in the coding phase of vstl.

 The PR10-1 inducible PMs promoter sequence includes 7 of the 13 nucleotides of the Ms PR10-1 gene sequence (in lower case in the box).

 The coding and non-coding parts (intron) of the portion corresponding to the sequence of the grapevine stilbene synthase gene are represented in upper and lower case letters, respectively.

Figure 6: Demonstration of gene induction coding for a stilbene synthase by U.V.

The RNAs are extracted from approximately 1 g of leaves 17 hours after UV induction. 10 to 20 μg are deposited on denaturing formaldehyde-formamide gel. After migration (3 V.cm1), the RNAs are transferred to nylon membrane and fixed by UV exposure (254 nm, 33 mJ.cm 2). The
Northern blot is obtained by hybridization at 650C overnight with the biotinylated vstl probe.

 The starting explant consists of isolated leaves of 41B (control) or 41B vitro-propagating plants transformed genetically with a construct (13 kb insert, comprising two complete stilbene synthase genes vst1, vst2, a piece of genomic vine DNA and another truncated stilbene synthase gene of vine (vst3) incorporating supernumerary copies of genes encoding vine stilbene synthases under control of their own promoters (clones 2 and 3 respectively corresponding to clones 55-2 and 55-3). 41B: Hybrid rootstock V. vinifera, Chasselas x V. berlandieri.

Figure 7: Kinetics of accumulation of stilbene synthase mRNAs after U.V. induction.

The RNAs are extracted from about 1 g of leaves. 10 to 20 μg are deposited on denaturing gel formaldehydeformamide. After migration (3 V.cm1), the RNAs are transferred onto a nylon membrane and fixed by exposure to
UV (254 nm, 33 mJ.cm2). Northern blot is obtained by hybridization at 650C overnight with the biotinylated vstl probe.

The explants are isolated leaves of 41B vitroplants. The control is a non-genetically transformed clone contrary to clones 2 and 3 (corresponding respectively to clones 55-2 and 55-3) which integrated into their genome the 13 kb insert (see above) containing genes coding for genes. stilbenes synthases of vines (vstl + vst2). 41B: hybrid rootstock V. vinifera,
Chasselas x V. berlandieri.

Figure 8: Quantities of resveratrol present in the sheets of vitroplants treated 8 min U.V. and analyzed at different periods after induction.

 The amounts are expressed in g per g of fresh material.

 NI: not induced.

 The control consists of leaves taken from unprocessed 41B. PCT 55-2 and 55-3 represent two transformants having integrated the 13 kb insert comprising two complete stilbene synthase genes (vstl and vst2).

Figure 9: Inhibition of growth of Botrytis cinerea mycelium, strain 916T, after 7 days at 200C.

 The culture of the mycelium is carried out on maltglucose medium containing the different concentrations of resveratrol.

Figure 10: Photo Board 1
Characteristic macroscopic observations of different vine varieties interacting with Botrytis cinerea 5 days after inoculation of vitroplant leaves with a suspension of conidia - Unprocessed plants.

Up
- Left: Mad white grape variety, 280 sensitive clone.

 - Right: Pinot noir grape, clone 386 moderately tolerant.

Below
- Left: Ugni-blanc grape, clone 479 tolerant.

 - Right: 41B rootstock tolerant.

Figure 11: Photo Plate 2
Characteristic macroscopic observations of clones of rootstock 41B, transformed by different constructs (145 PR10-1 promoter PMs - vstl 55 gene: 13 kb insert comprising two vstl and vst2 genes under control of their own promoters), in interaction with Botrytis cinerea , 5 days after inoculation of vitroplant leaves with a suspension of conidia.

Up
Left: Clone 145-2.

 - Right: Clone 145-5.

Below
- Left: Clone 145-6.

 - Right: Clone 55-3.

Figure 12: Photo Board 3
Characteristic observations, by fluorescence microscopy, of different vine varieties interacting with Botrytis cinerea, 5 days after inoculation of vitroplant leaves with a suspension of conidia.

 Fluorescence: filter block A (excitation from 340 to 380 nm, stop filter at 425 nm). Resveratrol emits fluorescent light in bluish white and blue (depending on its concentration), chlorophyll in red. The black zone corresponds to the zone of infection by the fungus or to necrotic zones when they are small.

Up
- Left: Mad white variety, 280 sensitive clone, little or no resveratrol synthesis.

 - Right: Pinot noir grape, clone 386 moderately tolerant. Synthesis of resveratrol in the veins and in the maceration zone of the fungus.

Below
Left: 41F (tolerant) rootstock not transformed. Synthesis of intense resveratrol in the veins and limb around small and very localized infection areas (necrotic areas).

 - Right: Rootstock 41B transformed by the construction 145, that is to say the promoter PMs PR10-1 vstl gene, clone 145-5 very tolerant. Strong synthesis of resveratrol around and, in the infection zone, in the veins and on almost the entire limb of the leaf.

Example 1
Obtaining csenomiped clones comprising querulous sequences of PR alfalfa protein molecules
A) Obtaining a probe for the search of promoters
(see Figures 1 and 1a)
The incompatible response (HR hypersensitivity reaction), obtained in the host-parasite alfalfa (Medicago sativa) and Pseudomonas syringae pv pisi relationship, made it possible to constitute a cDNA library. It was carried out from messenger RNAs extracted and purified from the neighboring area of the necrosis caused by the bacterial infection. Samples of plant material were taken 6 hours after infiltration with the bacterial suspension.

 In legumes, PR proteins are known to have conserved motifs, this allowed the synthesis of oligonucleotides corresponding to these patterns defined from sequencing already performed on PR pea and soy proteins. PCR amplification provided a radioactive probe, which was then used to select transcripts from the cDNA library.

Among these, one of the clones: cDNA-PR7 was retained because after sequencing it presented 87 W of homology with the genes coding for PR pea and soy proteins.

The analysis showed that it corresponds in fact to a gene encoding a PR class 10 protein according to the VAN LOON et al. (1994). It was named Ms PR10-1 (Medicago sativa PR protein class 10, clone 1).

A check, carried out in alfalfa by
Northern blot, showed that the corresponding transcript accumulated as early as 3 hours after infection in the case of the incompatible reaction, went through a maximum between 24 and 48 hours and decreased slowly from 72 hours.

This fragment is characterized by the existence of an internal Bam HI site (denoted B in FIG. 1) which delimits two parts
one so-called 5 ', of about 340 bases, includes the upstream region of ATG (which is transcribed but not translated) and a downstream sequence corresponding to 306 bases,
the other called 3 ', corresponds to the end of the coding part, ie 165 bases, and to the 3' untranslated region, ie 186 nucleotides from the stop codon to the beginning of the poly A.

B) Isolation of genomic clones comprising
Protein PR promoters
1) Isolation of genomic clones
A genomic library of alfalfa, prepared in
EMBL4 (title: 7.108 pfu (flat forming units) ml1), was used on this occasion and 6.105 pfu spread.

To screen this library, the 5 'fragment of Ms PR10-1 (cDNA-PR7) served as a probe and 45 clones gave an intense signal on 13 of them. The restriction maps and the hybridizations, carried out with the 5 'and 3' fragments of Ms PR10-1, made it possible to conclude the existence of 7 distinct clones (cf Figure 2). The comparison of the sizes of the 7 clones (9.3, 6.5, 6.1, 5.8, 4.8, 4.2, 2.2 kb) obtained from the screening of the library with that of the detected bands on alfalfa genomic DNA blot showed good agreement between these two types of experimental data (see Figure 2). it was therefore possible to deduce that the genes encoding the PR7 protein corresponded to a small multigene family.

 The fragments (EcoRI / EcoRI sites) of these clones (except for the C12 clone) were then subcloned in whole or in part and the sequencing carried out.

2) Sequencing performed
A clone was chosen to be sequenced first, clone C15 (see Figure 1a).

 The first sequencing work revealed the presence of an intron of approximately 315 nucleotides in the open reading frame of the gene encoding the PR protein. The clone studied was then analyzed after digestion with Bam HI (see FIG.

Clone C15: 6.1 kb
Cloning by Eco RI and Bam HI yielded two fragments EB (about 2.4 kb) and BE (about 3.7 kb). After sequencing and comparison of the coding sequence (interrupted by an intron of 600 nucleotides) with that of Ms PR10-1 (cDNA-PR7), it appeared that this genomic clone was absolutely identical to this reference cDNA.

3) Analysis of Expression of Isolated Clones in the Alfalfa-Pseudomonas System
The experiments focused on the C15 clone, using the 5 'extension technique, to determine the messengers actually transcribed during the induction of defense reactions. This technique also has the advantage of making it possible to locate the transcription initiation site. The results showed that the clone
C15 was actually expressed during the induction of defense reactions in the leaves, during alfalfa-Pseudomonas interaction.

Example 2
Genetic transformations performed to verify the promoter activity of the isolated clone
A) Promoting regions used
For these control transformations, two promoters were selected: a control promoter and the PR promoter isolated from the alfalfa genome, derived from C15 (corresponding to the PMs promoter PR10-1).

1) CaMV-35S Promoter
This promoter, called constitutive, is conventionally used. I1 is the transcriptional regulatory sequence of the cauliflower mosaic virus (CaMV) 35S RNA subunit gene. The promoter region which has been used to carry out the construction with the gus reporter gene corresponds in fact to a fraction of this promoter, again isolated in the form of a fragment.
EcoRI / BamHI from plasmid pDHS1 (PIETRZAK et al., 1986).

2) Promoter PR
The promoter region of the C15 genomic clone was studied. This promoter was later called PMs PR10-1.

PMs PR10-1:
It is derived from the 2.4 kb EcoRI / BamHI (E / B) fragment of clone C15 (Figure 1a). The integration of this fragment into the binary plasmid, upstream of the reporter gene (see below), was made difficult because no restriction site existed on the C15 clone between the TATA box (transcription initiation) and ATG (initiation of translation). Deletion experiments were thus performed until a fragment of about 1.5 kb (IND S1 sequence) was obtained. Then, by free-end ligation, another Bam HI site was added to the fragment thus obtained to allow its insertion upstream of the different coding sequences used thereafter. This fragment therefore comprises, with reference to the cDNA which served to clone it and in addition to the upstream promoter region: 39 terminal nucleotides of the 5'UTR (UnTranslated Region) of the Ms PR 10-1 gene, located 10 bp from the codon Initiator ATG, Ms PR10-1 gene ATG and a short fragment of its coding region (10 bp), just upstream of the integrated HI site.

Taking into account the cloning sites, the promoter thus constructed has a potential ATG which could lead to the presence of two ATG codons, at a short distance from one another, during the construction of chimeric genes. A risk of modification of the coding phase of the gene used (reporter gene or stilbene synthase gene) could then exist.

For transformation experiments with the reporter gene, PMs-PR10-1 (PRI) was used as is after cloning in the plasmid pSK +/- Bluescript of
Stratagene. It could be isolated again in the form of an EcoRI / BamHI fragment of about 1.5 kb.

3) Used plasmids
a) p35S - intron gus (VANCANNEYT et al., 1990)
This plasmid is a derivative of pBin19 (BEVAN, 1984) and has as such the right and left borders of the binary plasmids allowing the insertion of the part between these borders into the plants via agrobacteria.

 The development of the reporter gus intron gene (intron derived from the LSI gene of the potato) made it possible to eliminate false positives (in particular in transient expression) due to contaminating agrobacteria. They are unable to splice the introns.

Classically this gene, encoding a glucuronidase, allows, using a particular substrate (5-bromo-4-chloro-3-indolyl ss glucuronide), to obtain a blue color. This then indicates the transformed nature of the analyzed plant and consequently the transcription of the coding sequence of the gene corresponding to the enzyme, thus the induction of the promoter which controls it.
b) pPR97
This plasmid was constructed by one of the participating laboratories to test the efficacy of promoters (P. RATET, ISV, cited in SZABADOS et al., 1995). It has the particular advantage of having a multiple cloning site, allowing a transcriptional fusion with the coding phase of the gus gene (uid A of E. coli) containing the LSI intron. It also possesses some of the characteristics of the previous plasmid p35S-gus intron (integration borders in the plant genome, selection gene allowing resistance to neomycin-like antibiotics: npt II gene).

The promoter activity can be revealed and measured by histochemical and enzymatic tests of
GUS.

4) Derivatives of pPR97
Two main constructions were made and used later for the transformation of model plants.
a) pPR97 - 35S
The 35S promoter, cloned into plasmid pDH51 tcf. paragraph 5 below), was extracted and inserted into the multiple cloning site of pPR97, upstream of the reporter gene, as an EcoRI / BamHI fragment. This plasmid is both a positive control, to demonstrate that the construct is functional, and a reference because the 35S promoter has been placed in the same environment as the promoter isolated from PR protein clones.
b) pPR97-PMs PR10-1
The 1.5 kb were inserted into the same cloning site as defined above, again in the form of an EcoRI / BamHI fragment.
c) pG3-3
It allowed for a strong positive control for histochemical and enzymatic assays by cloning two 355 inverted tandem promoters. The activator sequences of the promoters then act in synergy. The coding phase of the gus intron gene was then placed under the control of one of the two 35S promoters.

5) Strains of agrobacteria carried out
The different plasmids were used to transform E. coli strain DH5a competent bacteria by heat shock in calcium chloride medium. After selection of bacteria transformed on antibiotic medium (Kanamycin) and miniprep control of their recombinant nature, they were used to transfer the recombinant plasmids into strains of Agrobacteria by triparental conjugation, using the strain of E. coli. coli
HB101 containing autotransferable plasmid pRK2013 (DITTA et al., 1985).

Two strains of agrobacteria were retained
EHA 105, Agrobacterium tumefaciens disarmed, allowing the regeneration of transformed plants (stable transformations), and A4TC24, Agrobacterium rhizogenes used to obtain the reaction of hairy root type and composite plants, having transformed roots but whose rest of the plant is identical in phenotype to that of origin.

6) Genetic transformations on model plants
Two types of transformations (transient and stable) were made using three model plants Nicotiana benthamiana, Medicago truncatula and Lotus corniculatus.

 The results obtained with N. benthamiana will be mainly exposed.

7) Transitional transformations
This first series of experiments was performed to allow a quick verification of the functionality of constructs made with the gus gene in eukaryotic cells.

 Leaves of N. benthamiana were excised and cocultured on agar medium with the various derivatives of the strain EHA 105. Histochemical tests were then performed 48 hours after the transformation and then examined after a night of incubation (12 h ).

 The p35S-gus intron and pPR97-35S control plasmids gave a positive but weak GUS staining with the second plasmid.

 This low reactivity probably comes from a construction problem because in the vicinity of the transcription initiation site and the gene ATG part of the polylinker had to be kept. This having a repeated sequence may interfere with the transcription of the gene.

 The PR10-1 pPR97-PMs construct showed gus gene activity similar to that of the positive control 35S-gus intron. This promoter, which was expected to be inducible, therefore had an effect comparable to a constitutive promoter.

 This result can be explained as the consequence of either bacterial infection or injury to the leaves during harvesting or culturing. Since the first hypothesis can not be verified, the experimental protocol was modified to reduce the stress caused to the explants (elevation of the osmolarity of the coculture medium by using sucrose concentrations of 10 to 30 gl -1, making a range of vacuum more or less pushed: 10 to 80 mm of mercury relative vacuum and creation of more or less strong lesions on the leaves with heavy or medium crushing of the epidermis).

 The results showed that the higher the pressure, the higher the number of transformed cells. However, a compromise must be found to obtain stable transformations because the many transient transformations, obtained in this case, are subsequently often shown to be lethal for the cells.

They are then unable to give callus and thus regenerate buds and plants. Moreover, the inducible nature of the promoter is partly confirmed because if the coloration due to the 35S-gus intron construct is detectable up to 5 days after coculture, that obtained with pPR97-PMs PR10-l-gus intron appears more rapidly at 48 hours but then decreases very quickly.

8) Stable transformations
a) Tobacco: N. benthamiana
A series of transformations were carried out with pPR97-35S, pPR97-PMs PR10-1 and pG3-3. A large number of seedlings were obtained for this series of transformations. For each of the plasmids used, we sought to obtain at least 7 plants acclimated.

However, this was not possible for p35S-gus intron or only. 5 plants were regenerated and acclimated.

The results obtained are collated in Table 1.

Table 1: Stable transformations obtained on N. benthamiana by transformation with the strain EHA 105 of Agrobacterium tumefaciens and its derivatives.

<Tb>

<SEP> Cals / explants <SEP> Buds <SEP> | <SEP> Seeds
<tb><SEP> Constructions <SEP> mis <SEP> in <SEP> obtained
<tb><SEP> culture <SEP> in <SEP> vivo <SEP> Accl.
<tb> p35S-gus <SEP> intron <SEP> 10 (45) <SEP> 6 (1) <SEP> 5 <SEP> 5 <SEP>
<tb><SEP> pPR97-35S <SEP> gus <SEP> 115/122 <SEP> 23 <SEP> 13 <SEP> 12
<tb><SEP> intron
<tb> pPR97-PMs <SEP> PR10-1 <SEP> 135/139 <SEP> 27 <SEP> 20 <SEP> 7
<tb><SEP> gus <SEP> intron
<tb><SEP> pG3-3-35S <SEP> in
<tb> tandem <SEP> inverted <SEP> no <SEP> evaluated <SEP> 37 <SEP> 28 <SEP> 20
<tb><SEP> (promot. <SEP> Strong)
<Tb>
Legend of Table 1
The results are expressed in quantity obtained.

Acc. : acclimated seedlings.

Number of buds per explant: the number in parentheses corresponds to the number of buds obtained at one month, for the first series. For this one (with the construction 35S gus intron), the calli were left longer on the culture medium to obtain a maximum of buds and therefore plants to acclimate. For the second series, after one month, a sufficient number of buds were obtained and the experiment was then stopped.

Genetic transformations: they are classically made on leaf blade pieces of 1 cm 2, immersed for 30 seconds in the Agrobacterium suspension and then co-cultured for 48 hours before transplanting into agar medium of cell division and caulogenesis (MURASHIGE et al. 1962), ANA (naphthalene acetic acid) 0.1 mg.l 1, BAP (benzyl amino purine) 1 mg.ll, cefotaxime 400 mg.ll (elimination of agrobacteria) and kanamycin 70 mg.ll (cell selection agent transformed).

As soon as the first buds appear (approximately one month after coculture), they are placed on rooting medium identical to the first but not including plant hormones.

 The rapid analysis of the results, according to the expression of the gus intron gene according to the nature of the promoter situated upstream of the coding phase of the gene, shows that the PMs promoter PR10-1 gives the best results of all the promoters. tested. A more detailed analysis is presented below.

9) Characteristics of PMs promoter PR10-1
Plasmid pPR97-PMs PR10-l-qus intron
This promoter gave the best results with differences in constitutive expression of the gus gene according to the organs tested.
a) Activity in the calluses
Strong constitutive expression was found.

A few minutes of incubation were sufficient to obtain a positive histochemical test. The promoter is therefore strongly induced in this type of material, which is in agreement with the results obtained by VAN LOON (1985). The calli in vitro culture are in a state of stress and under these conditions the PR proteins are expressed.
b) Activity on whole plants acclimated
roots
The promoter is induced and the histochemical test is positive after 2 hours of incubation (against 5 hours with the 35S promoter). The activity of the gus gene is not uniform in the tobacco roots, only the epidermis of the elderly parts and the apical meristem gave the characteristic blue color of the test. According to the literature an activity of defense genes in the roots is also conventionally observed.

flowers
In all the tobaccos transformed by this construction, a strong constitutive activity was found in the flowers and more particularly in the anthers and the pollen. Gus gene activity was also detected in the trichomes of the sepals and more weakly in the petals. These results are also in agreement with those of
VAN LOON (1985) who indicate an induction of defense genes in floral pieces.

leaves
Low constitutive activity was observed in trichomes of young leaves of adult plants. In tobacco, the broadleaf rosette stage corresponds to the juvenile stage and the aging stage to that of seed formation. This low activity was found mainly in multicellular trichomes. The expression of a PR protein in such structures has never been described to our knowledge.

 An inducing activity constitutive of the isolated PRLO-1 PMs promoter is thus possible in the trichomes of tobacco leaves (3 plants out of 7) but it seems to be influenced by the stages of development.

 In the absence of pathogen induction, promoter activity in tobacco. is therefore limited to the root, floral pieces (anthers and pollen) and some cells of the aerial part (trichomes essentially).

Example 3
Other transformed plant species 1) Medicago truncatula
For this species, attempts have been made to produce composite plants, that is to say having both a wild-type (non-genetically modified) aerial part and transformed roots.

 Young sprouts were used. After development of the main root, the excised hypocotyls were dipped in a suspension of Agrobacterium tumefaciens EHA 105 possessing either the plasmid p35S-gus intron or the plasmid pPR97-PMs PR10-1gus intron, so as to obtain subsequently transformed neoformed roots. After a week, roots were obtained and a GUS histochemical test was performed.

For this experiment, the control was constituted by young germinations treated identically to the previous batches (hypocotyls excised but not soaked in the suspension of agrobacteria).

 All of the explants in the control group had rooted in a week, however, only 50 k reacted for the Agrobacterial treated groups. Regardless of the treatment, no root gave a positive response to the GUS test. In contrast, the hypocotyl base of the treated lots, although necrotic, often reacted to give a blue color (presence of transformed cells). The necrotic part of the explants was then excised and they were re-rooted. 50 k then developed neoformed roots some of which were found, in some areas, positive test.

 Thus, chimeric roots (transformed and untransformed cells) were obtained, the transformed portions corresponding to cell lines having integrated the construct into a basic stem cell.

 The two constructs tested, p35S-gus intron and pPR97-PMs PR10-1-gus intron, yielded these transformed root cell lines in respectively 3 and 2 explants out of 6 tested for each lot.

Although the experimental model is not adapted to the study pursued (study of the expression of PR proteins during the phenomenon of nodulation by
Rhizobium), it has nonetheless shown that the sponsor
PMs PR10-1 is just as functional in this plant as in the original plant (Medicago sativa).

2) Lotus corniculatus
The purpose of the experiment was also to study the induction of the promoter during nodulation by the symbiotic bacterium Rhizobium meliloti NZP 2037 (PETIT et al., 1987). Composite plants were thus made by transforming hypocotyl cells of young Lotier germinations by Agrobacterium rhizogenes strain A4TC24, to obtain the phenomenon of root hair (hairy root phenotype). Once this developed, the main roots were excised and the seedlings were placed in liquid medium to increase the development of the phenomenon. Once the plants acclimated, the study of the induction of promoters during the nitrogen-fixing symbiosis was carried out by placing the seedlings under nodulation conditions (BLONDON, 1964). The two study proponents retained were the same as those used during the experiment conducted with
M. truncatula: 35S and PMs PR10-1. For these two constructs, less than 10 k of hairy root roots showed GUS positive roots. In general, roots with this phenotype gave less nodules than control roots.

 For those obtained with the 35S promoter construct, only the nodules showed a positive response to the GUS test, while for the other (PR10-1 PMs promoter), the staining developed throughout the root, except at the point of initiation of secondary roots. Moreover, this last construction did not make it possible to obtain nodules on the roots resulting from root hair in interaction with Rhizobium meliloti.

Example 4
Study of the hypersensitivity reaction of transformed tobacco with the constructs using promoters of the alfalfa PR gene and the bolus pus intron
A) Hypersensitivity reaction on N. benthamiana
transformed with constructions associating the
alfalfa PR gene promoter and the gene sus intron
1) Hypersensitivity reaction test (HR)
The hypersensitivity reaction (HR), developed in the interaction N. benthamiana - Pseudomonas syringae pv. pisi, was used in this study. Transformed tobacco plants having incorporated in their genome the different inserts of plasmids p35S gus intron and pPR97
PMs PR10-l-gus intron, were acclimated and then infiltrated by a suspension of P. syringae (ESNAULT et al., 1993) at a concentration of 109 bacteria per ml. The solution was injected into the limbus using a hypodermic syringe. With such a model, in 48 hours, the HR type reaction is considered well developed. The leaves, infiltrated by the bacterial suspensions, were taken 24, 48 and 96 hours after inoculation to evaluate, by GUS histochemical test, the induction of the different promoters studied. The analysis of a possible systemic response was also evaluated, using the same histochemical test, on leaves below the infiltrated leaf.

2) Study of the induction of promoters under HR-type reaction conditions
a) Constitutive promoter 35S
In the case of the 35S constitutive promoter (plasmid pG3-3 for example), the inoculation with P. syringae did not modify the test response and this in a few minutes of incubation. The infiltration by the bacteria does not therefore alter the constitutive type glucuronidase activity obtained with the 35S promoter.
b) Protein gene promoters PR
PMs PR10-1 Promoter
As shown in Table 2, this promoter is well inducible by attack by a pathogen. At 24 hours, the HR-type reaction is not yet fully developed (48 hours for complete exposure), however, the GUS test is already positive. With the transformed young tobacco plants obtained, the coloring is weak and produced mainly in the limb of the infiltrated leaf.

 In systemic response, response at the level of the lower leaf to the infected leaf, staining is only in limbo. The adult tobacco plants (stems developed but not yet flowering) and juveniles (in rosette) have the same type of response with a weak activity of the gus gene, determined by the histochemical test.

 For older, flowering or seeded tobaccos, the staining obtained during the test is more intense, especially in the veins and trichomes of the infected leaf and only in these tissues for the systemic response.

Differences in the expression of the reporter gene are therefore demonstrated according to the age of the plant. This response, which depends on the stage of development of the plant, has been found in most studies of plant PR proteins. Induction of PM promoter
PR10-1 is a transient phenomenon since the expression of the reporter gene is no longer visible 96 hours after inoculation by bacteria.

 Induction is also not limited to the HR type reaction obtained during the plant / bacteria interaction. The same type of response was obtained with PMs PR10-1-gus intron construction during an infection of one of the explants by a fungus. A homogeneous expression of the gus gene was then visible on the whole of the infected leaf, except the zone of contamination which itself was necrotic.

Table 2: Induction of the different promoters studied during the HR type reaction between N. benthamiana and P. syringae.

<Tb>

<SEP> 24h <SEP> after <SEP> inoculation <SEP> 96h <SEP> after <SEP> inoculat.
<Tb>

Construction <SEP> Sheet <SEP> Sheet <SEP> sheet <SEP> infiltrated
<tb><SEP> infiltrated <SEP> lower <SEP> reaction <SEP> type <SEP> HR
<tb> PMs <SEP> PR10-1 <SEP> 6/7 <SEP> 6/7 <SEP> 0/3
<tb> pG3-3 <SEP> (35S) <SEP> 3/3 <SEP> 3/3 <SEP> 2/2
<Tb>
Legend of Table 2
The results are presented in number of plants responding positively to the GUS histochemical test in relation to the number of plants analyzed.

B) Quantitative expression of the above-mentioned intron gene
control of the different promoters
The method is based on an enzymatic test.

She uses
a) a crude extract of the enzyme encoded by the gus gene obtained from the transformed tobacco plants, and
b) a substrate, p-nitrophenyl glucuronide. The rate of hydrolysis of the substrate is monitored spectrophotometrically and is related to the total amount of protein in the extract. The method requires the presence of a strong promoter, upstream of the gus gene, because it is insensitive.

 As a result, it was not applied to tests for which 12 hours or more of incubation with the substrate, used for the histochemical test (X gluc: 5bromo - 4 - chloro - 3 - indolyl - ss glucuronide), were required.

Under these experimental conditions, the 35S promoter, placed in the plasmid pG3-3, gave a substrate hydrolysis rate (expressed in arbitrary units) 5 times higher than the PMs PR10-1 promoter, placed in the plasmid pPR97. On the other hand, the PMs promoter
PR10-1 gave a higher expression of the gus gene than the 35S promoter, if it is placed in the plasmid p35Sgus intron. Indeed, in the latter case, no detection of hydrolysis of the substrate was obtained.

 Likewise, the other constructions did not make it possible to give detectable values to the spectrophotometer.

Example 5
Isolation of DNA corresponding to a svanthase stilbene vene and expression of stilbene synthase
Two methods were used to obtain a gene encoding a vine stilbene synthase. On the one hand, data from the literature (WIESE et al., 1394) made it possible to know the sequence of a gene. On the other hand, a genomic insert of about 13 kb was provided by
BAYER AG (Agrochemical Division Research / Biotechnology
Pflanzenschutzzentrum, MONHEIM, D-51368 LEVERKUSSEN). It is specified that the BAYER company has filed with the
Deutsche Sammlung von Mikroorganismen (DSM), in Germany, strains of E. coli containing plasmids carrying vine stilbene synthase genes (see EP-464 461): E. coli strain Fier 1 pVst 1 (DSM 6002, filed June 18, 1990), E. coli strain Fier 2 pvst 2 (DSM 6003, filed June 18, 1990), and E. coli Fier pvst 1 2 t 3 strain (DSM 6346, filed February 11, 1991). Bayer has also deposited E. coli strain Nurdug 2010 (DSM 4243, filed September 17, 1997) which contains the plasmid pGS 828.1 which carries a peanut stilbene synthase gene (see EP-309 862). The insert used to carry out the work reported below corresponds in fact to a complex genomic clone comprising two complete functional sequences of stilbene synthase genes (vstl and vst2 genes) and an incomplete sequence vst3. Subsequently, the sequence of the vstl gene was selected to be incorporated into the realized constructs. It corresponds to a genomic fragment of 4.9 kb (functional sequence including the promoter) which does not have suitable restriction sites to allow direct cloning in the plasmids usually used as transformation vectors. Complementary sites were thus added by intermediate cloning in a plasmid pCDNA II.

A) Alone complementary sites by cloning
intermediate in a plasmid pCDNA II
The genomic fragment of the already indicated vst1 gene was isolated from the original plasmid in which it had been cloned as an EcoRI / PstI fragment (2.1 kb) and cloned again into a pUCI9 plasmid. Once cloned, the vst1 fragment was incorporated into the same sites (EcoRI / PstI) of plasmid pCDNA II to change the restriction sites, to remove the terminator of the gene and to allow the isolation of a BamHI / BamHI insert (1, 8 kb). It is this fragment, corresponding to the open frame of reading of the gene, which was used to make the constructions with the different promoters including those isolated from the genomic bank.

B) Study of the expression of the vstl gene
To verify the expression of genes encoding stilbene synthase in vine plants, a probe (1.8 kb), including the vsti gene, was prepared from plasmid pCDNA II multiplied in E. coli Hs bacteria. The probe was then biotinylated by random priming, with the Polar Plex kit (Plex chemiluminescent kits,
Millipore), to allow detection of genes or transcripts coding for a stilbene synthase by chemiluminescence on Southern and Northern blots, made from nucleic acids extracted from control or transformed vine plants.

1) Use of the vstl probe for the analysis of genomic DNA extracted from vine 41B (Hybrid V. vinifera
Chasselas x V. berlandieri; rootstock)
Southern blot analyzes were performed after extraction of genomic DNA and then digestion thereof with EcoRI. The vstl probe made it possible to obtain a large number of bands (approximately 15). These correspond in fact to fragments containing sequences coding for a stilbene synthase which constitute a multigene family (6 to 8 genes according to WIESE et al., 1994). Of these, vsti, vst2 and vst3 are highly homologous to each other. In addition other genes can be recognized by this probe, including those corresponding to the multigene family of chalcone synthetase. The two enzymes are indeed of the same structure and of the same size (subunit dimers from 41 to 44 kb). They also use the same substrate and their amino acid sequence has a high degree of homology, at least at the active site.

 To verify whether such cross-hybridization was possible, the DNA of two plasmids was extracted.

One, pCDNA II, contained the vstl gene, and the other, pPCV 002, a Rosier chalcone synthase gene, available in the laboratory. A biotinylated probe corresponding to the Rosier chalcone synthase gene was also prepared. The
Southern blots obtained, carrying out the cross hybridizations, showed that the vstl probe actually recognized the Rosier chalcone synthase fragment and vice versa. The signals emitted are then weaker in this case of cross hybridization.

2) Determination of resveratrol from leaves of vine plants
The fresh plant material, leaves or stems, is reduced to powder in a mortar containing liquid nitrogen and the apolar compounds are extracted with methanol (1 ml per 100 mg of fresh material: mf).

 After centrifugation to remove debris, the methanolic extract is filtered through a 0.45 μm filter and then evaporated to dryness under nitrogen. The residue is taken up in pure methanol (100 μl / 100 mg m.F.). In order to eliminate the pigments (chlorophylls in particular), the sample is then passed through a C18 column (Sep-pack Waters), previously equilibrated with methanol. The qualitative and quantitative analysis of the extracts is carried out by C.L.H.P.

(High Pressure Liquid Chromatography) on a C.L.H.P.

WATERS (Model 600 E), coupled to a diode array detector (Model 990. WATERS). The chromatography support is composed of a reverse phase C18 column (ultra-basic
C18, 205 x 4.6 mm, 5 Hm; Shandon). The analysis of the compounds of the extract is carried out under isocratic conditions, the mobile phase consisting of a mixture of acetonitrile-water 35/65, V / V with a flow rate of 1 ml.min-1.

 An absorption spectrum is made every two seconds between 200 and 400 nm and resveratrol is detected at its maximum adsorption at 305 nm.

 The quantification of resveratrol is carried out by external calibration from a standard straight line, obtained by chromatography of solutions at 5, 10, 20, 50 and 100 μg / ml, made from commercial resveratrol (Sigma). The resveratrol concentration, measured from the peak area corresponding to the molecule, is referred to the mass unit of m. f. or of m. s. (dry matter) or chlorophyll mass of the sample dose.

Example 6
Nucleic acid constructs associating the gene encoding a stilbene svnthase from vine to different promoters 1) Constructs using constitutive promoters
Two related promoters have been used to carry out genetic constructs to allow constitutive expression.

In the first construct, vine stilbene synthase (vstl) DNA was placed under control of a regulatory sequence consisting of a cassette containing two CaMV 35S promoters mounted in series (in the same orientation). At the end of the coding sequence of the vst1 gene, a 35S polyadenylation sequence was also added. The chimeric construction thus produced can be summarized as follows: p35S (CaMV) - p35S (CaMV) - vstl - poly
At 35S (CaMV).

In the second construct, the vst1 coding sequence was placed under the control of four enhancer sequences isolated from the CaMV 35S promoter and, serially placed in front of the CaMV 35S promoter and the vine gene promoter (vstl) enhancer sequence.
The two chimeric sequences thus produced were first inserted into the plasmid PMP 90RK and then the plasmids were subsequently incorporated into the Agrobacteria strain GV3101.

 These two regulatory sequences are considered as strong constitutive promoters.

2) Homologous construction using the 13 kb insert
(vst genes under control of their own promoters)
The approximately 13 kb fragment of genomic vine DNA has been described above. It includes 2 functional genes (vstl and vst2) coding for stilbene synthase enzymes and an incomplete (non-functional) gene (vst3). This vine sequence was co-integrated with a plasmid pGV3850 which was then introduced into a strain of agrobacteria. It thus corresponds to open frames for reading vst genes (vine stilbene synthase) under the control of their own promoters.

 Several series of plants 41B, transformed by the plasmid comprising the 13 kb fragment, were obtained and were studied and analyzed by Southern blot using 3 different probes (1.8 kb probe of the nptII gene, 2.4 kb of the ainpicillin resistance gene and 1 kb of the left border of plasmid pBIN 19 comprising the end of integration sequence of TDNA). The majority of the selected plants reacted to one or other of these probes.

These clones were numbered according to a code: 55 for the construction and 2, 3, 5, 6, 7, 9 for the various transformants obtained.

3) Constructs using the PM inducible promoter
PR-10-1
The construct comprising the PR10-1 PMs promoter, isolated from alfalfa PR genomic clones, was performed (see Figure 5).

Construction of plasmid pBin 19 - PMs PR-10-1 - vstl gene
35S Terminator
The PMs PR10-1 promoter isolated from alfalfa and the vstl gene each having a translational ATG codon, an adapter was made to clone the vstl gene without additional ATG in the construct. This adapter was synthesized in the form of two oligonucleotides of 11 bp each, one compatible Bam HI, the other Mun I compatible. The adapter was then incorporated into the Mun I site of the vst1 gene cloned into plasmid pUC19. The insert was then recovered by Bam digestion
HI, then cloned into pBIN 19 between the PR10-1 PMs promoter and the 35S terminator. The insertion of the vstl gene with its adapter therefore lies between the PMs PR10-1 promoter and the 35S terminator. Under these conditions, the ATG of the vst1 gene was eliminated and 3 additional codons were included in the coding phase of vst1, upstream of the gene. It was verified by sequencing that the open reading frame of the gene was still in phase with the rest of the construct.

 After transformation of the 41B plants, with the insert comprising among others the chimeric promoter sequence PMs PR10-1-vS1 terminator 35S gene, the transformants were analyzed by Southern blot, using the nptII probe already described. Nevertheless, complete integration can not be fully demonstrated by this method and especially by this probe, since in the Agrobacteriuin system it is accepted that the insertion into the genome of the plant begins at the right border and ends at the from the left. Since the nptII gene is located in the construction used near the right border, a partial integration, nptII gene inserted but subsequent blocking of the integration before the left border, is possible. Transformants were coded 145 and assigned numbers 2, 5, 6 for those whose results are shown below.

Example 7
Genetic transformation of the vine and analysis of the effectiveness of the promoters studied
As described above (Example 6), four major constructs were made to transform the model system of the 41B rootstock laboratory (V. vinifera x V. berlandieri). The latter has the advantage of giving good results in transformation of embryogenic cell suspensions by agrobacteria.

About 50 transformants are obtained on average by experimentation using 0.1 to 1 μl of PCV (Packed
Cell Volume) of embryogenic cells. In addition, selection, development of transformed embryos and regeneration into plants are fast. In vitro seedlings with 6 to 8 well-developed leaves can be obtained in two months of culture.

A) Genetic transformation of 41B with vectors
having the vstl gene under promoter control
constituent
Two constructs were tested, one comprising the double 35S promoter mounted in series in front of the vst1 gene and the second consisting of a regulatory sequence composed of 4 35S enhancer sequences (CaMV) connected in series in front of the 35S promoter of CaMV. all located upstream of the vstl gene having its own enhancer sequence. Four series were conducted (two for each construct) to transform embryogenic vine cells. None has regenerated plants or even embryos. In all cases, rapid necrosis of embryogenic cell suspensions was obtained after 48 hours of co-culture with agrobacteria. These constructs do not therefore make it possible to obtain transformed plants that constitutively express the vstl gene under the control of these strong promoters. One hypothesis may be advanced, the expression of this gene could block the regeneration in embryos by rapid necrosis of cells with embryogenic potential in response to the production of stilbene phytoalexins.

 These negative results, obtained with the constructions comprising constitutive promoters of the 35S type, demonstrate the interest of controlling an overexpression of the vstl gene by a homologous or heterologous promoter inducible, in particular by a pathogen.

 These results are similar to those published by FISHER and HAIN in 1994, which highlight the fact that they failed to constitutively express a groundnut stilbene synthase gene at a high level in tobacco, using the 35S promoter of CaMV. According to these authors, there would be, with such a construction, a negative regulation of the expression of the gene in pathogen attack condition. This, according to their hypothesis, could result from the establishment by the plant of defense mechanisms, normally induced by pathogens (synthesis of PR proteins in particular) which would exert an inhibition on the viral promoters.

B) Genetic transformations of 41B by vectors
before the vstl gene under promoter control
inducible by abiotic and / or biotic stress
1) Study of the Expression of the Vst Genes and Their UV Induction Kinetics on Excised Leaves, Isolated in Survival and on Whole Plants of Control 41B Vine Vitroplants Controlled or Transformed by the 13-kb Insert
It has been shown that the synthesis of resveratrol (phytoalexin main grape), produced by the reaction catalyzed by a stilbene synthase enzyme, was inducible by ultraviolet (UV), therefore under conditions of abiotic stress (LANGCAKE et al., 1977
SBAGHI et al., 1993). Under normal conditions, the vine does not synthesize or very little resveratrol, on the other hand after UV induction (exposure of 10 minutes to UV at 254 nm, for a dissipating lamp 600 tiW.cm2), followed by a period of 20 hours In the dark, phyto-alexin is synthesized in the excised leaves.
a) Method used
Irradiation of leaves - tissue of vitroplants: at 254 nm for 13 minutes
for a dissipating lamp 600 CLW.cm-2, - leaves of vitroplants or vitroplants: at 254 nm
for 8 minutes for a dissipating lamp 600 UW.cm ~ 2, - extraction and analysis of the mRNAs of the plant material and
resveratrol estimation by fluorescence after
excitation at 365 nm at a given time after induction.

 Each sample is constituted by 3 isolated leaves of the same plant, induced separately to the U.V. but united for the extraction and the dosage. The test is performed on excised leaves, isolated from vitroplants, or on vitroplants in culture on agar medium, having 6 to 7 well developed leaves. The three oldest leaves of each seedling are taken and used for the test. U.V. exposure is performed on their upper side. After analysis, the amounts of resveratrol obtained are expressed in Fg of product reported either per gram of fresh weight of the analyzed leaves, or per gram of dry matter (evaluated on the pellet, after centrifugation after the methanolic extraction) or in mg of resveratrol per g of chlorophyll.

In the tables below, the values obtained for 41B, 55-X clones, transformed by the construction of the 13 kb genomic clone, where are present, in the sequence used as insert, two complete genes vstl and vst2. both genes under control of their own promoters.
b) Abiotic stress (UV) study on excised sheet (experiment N "1)
The results, corresponding to construct 55 (insert 13 kb) and clones 2 (55-2) and 3 (55-3), are shown in Tables 3 and 4 below (resveratrol assay) and represented by the Figures 6 and 7 (transcript analysis). The study of induction kinetics by
UV expression of the genes encoding stilbene synthase was performed after a period separating the UV induction of the analyzes fixed at 17 hours (see Table 3 and Figure 6), or varying from 0, 8, 17, 24 or 32 hours after induction (see Table 4 and Figures 7 and 8).

Table 3: Quantities of resveratrol detected in the control and transformed leaves, not induced or 17 hours after UV induction (expressed in CLg.gl of fresh material)

<tb><SEP> Controls <SEP> no <SEP> Clone <SEP> 55-2 <SEP> Clone <SEP> 55-3
<tb><SEP> transformed
<tb><SEP> No <SEP> Induced <SEP> No <SEP> Induced <SEP> No <SEP> Induced
<tb> induces <SEP> induces <SEP> induced
<tb><SEP> 0 <SEP> 12 <SEP> 0 <SEP> 11 <SEP> 0 <SEP> 13
<Tb>
Legend of Table 3: The leaves were excised from 41B tissue culture plants before UV induction. Clones 552 and 55-3 correspond to transformed plants having integrated a 13 kb insert containing genes encoding stilbene synthase.

 The fluorescence, observed in blue-violet at about 450 nm, is very strong in the veins of the excised leaves and evenly distributed over the entire surface of the leaf. Control leaves, which are not U.V. induced, do not exhibit fluorescence. These results highlight a specific expression of genes encoding stilbene synthases. The Northern blot transcrits analysis, performed by the vstl probe, shows the presence of a fragment of about 1.8 kb, the emitted signal of which is very intense in the UV-induced leaves, whereas it is absent or very weak. low in uninduced plants (cf.

Figure 6).

Table 4: Kinetics of the evolution of resveratrol concentrations on 41B sheets of UV-induced tissue culture taken from the plants in culture on agar medium, then isolated and analyzed for their resveratrol content at different periods after induction.

<Tb>

Time <SEP> after <SEP> Quantity <SEP> of <SEP> Resveratrol
<tb> induction <SEP> (h) <SEP> (in <SEP> freeze <SEP> of <SEP> Matter <SEP> Fresh)
<tb><SEP> Control <SEP> PCT <SEP> 55-2 <SEP> PCT <SEP> 55-3
<tb><SEP> NI <SEP> O <SEP> 0 <SEP> 0
<tb><SEP> 0 <SEP> 0.6 <SEP> 0 <SEP> 0
<tb><SEP> 8 <SEP> 30.8 <SEP> 12.1 <SEP> 9
<tb><SEP> 17 <SEP> 56.8 <SEP> 71.7 <SEP> 47
<tb><SEP> 24 <SEP> 83.2 <SEP> 81.4 <SEP> 35.2
<tb><SEP> 32 <SEP> 15.5 <SEP> 7.7 <SEQ> 151.8
<tb> Table 4: The results are expressed in yg. gl of fresh material. Two transformed plants PCT 55-2 and PCT 55-3 were analyzed in comparison with the control.

NI: Not induced at U.V ..

 Under these conditions, after U.V. stress, the amount of resveratrol found in the control leaves is maximum 24 hours after induction (of the order of 80 Ugg-1 m.f. for 41B). However, there are significant variations depending on the date of the analysis in the transplanting cycle of vitroplants: microbouturing cycle.

 The results obtained after Northern blot analysis, using the vstl probe, are shown in FIG. 7. At least two types of transcripts, of very similar size (approximately 1.8 kb), were detected. Although cross-hybridization with a chalcone synthase tanscrit is not excluded, the different recognized messenger RNAs probably correspond to the expression of different stilbene synthase genes. It has indeed been shown in the vineyard (WIESE et al., 1994, KINDL1 personal communication) that differences existed in the kinetics of induction of the different genes of the multigene family coding for stilbene synthases.

 Northern blot analysis revealed that in excised vine leaves 41B, which are then subjected to the abiotic stress of UV exposure, stilbene synthase transcripts exhibit a maximum of expression approximately 17 hours to 32 hours after induction.

These results are comparable to those obtained with vine cell cultures, which also showed the same pattern of expression but with the presence of two maxima (WIESE et al., 1994).
c) Study of abiotic stress (UV) on leaf isolated in survival (experiment NO 2), induction on vitro-plants before isolation of leaves
Table 5 below presents the results obtained for a second series of transformants having integrated the 13 kb insert. The study of the UV induction kinetics of the expression of the genes coding for stilbene synthase was carried out after a period separating the UV induction of the analyzes varying from 20, 40 or 60 hours.
Table 5: Resveratrol concentration of leaves isolated from vitroplants of vines after UV induction and extraction at different survival times.

<Tb>

<SEP> Time <SEP> of <SEP> survival <SEP> of <SEP> isolated <SEP> leaves <SEP> of
<tb><SEP> tissue culture <SEP> after <SEP> induction <SEP> at <SEP> UV <SEP> (8mn <SEP> to
<tb><SEP> 254 <SEP> nm) <SEP> and <SEP> before <SEP> extraction <SEP> of <SEP> resveratrol
<tb> Clone <SEP> studied <SEP> 20 <SEP> hours <SEP> 40 <SEP> hours <SEP> 60 <SEP> hours
<tb> 41B <SEP> - <SEP><SEP> Indicator o <SEP> 0 <SEP> 0
<tb><SEP> no <SEP> induced
<tb> 41B <SEP> - <SEP> Control <SEP> 281 <SEP> 165 <SEP> 6
<tb><SEP> induced
<tb><SEP> Clone <SEP> 55-2 <SEP> 135 <SEP> 918 <SEP> 58
<tb><SEP> induced
<tb><SEP> Clone <SEP> 55-3 <SEP> 44 <SEP> 931 <SEP> 301
<tb><SEP> induced
<tb><SEP> Clone <SEP> 55-5 <SEP> 392 <SEP> 83 <SEP> 657
<tb><SEP> induced
<tb><SEP> Clone <SEP> 55-6 <SEP> 339 <SEP> 235 <SEP> 43
<tb><SEP> induced
<tb><SEP> Clone <SEP> 55-7 <SEP> 263 <SEQ> 411 <SEP> 148
<tb><SEP> induced
<tb><SEP> Clone <SEP> 55-9 <SEP> 386 <SE> 823 <SEP> 54
<tb><SEP> induced
<Tb>
Legend of Table 5
The resveratrol concentrations are expressed in μg of dry matter.

 41B hybrid rootstock V. vinifera Chasselas x V.

Berlandierri.

The results presented in Table 5 distinguish two groups of plants - the first corresponds to the control and two of the
transformants 55-6 and 55-7. They usually have a
maximum concentration of resveratrol between 280
and 410 Lg.g 1 dry matter and this in general 20 hours
after induction except for clone 55-7 where it is located
at 40 hours - the second group presents meanwhile maxima more
high is almost double the first (820 to 930 ptg.
dry matter). It consists solely of
transformants (55-2, 55-3 and 55-9). The maximum is
expressed 40 hours after UV induction, however,
20 hours after induction, these plants often have
concentrations much lower than those of the first
group. This is the case for example clones 55-2 and 55-3
(135 and 44 Cig.gl dry matter respectively). Mon
clones, 55-9, is however interesting since
shows resveratrol concentrations greater than
those of the control in all cases (386, 823 and 54 gg-l
dry matter for 20, 40 and 60 hours
after induction).

* In this surviving leaf system, an uninduced control never shows resveratrol and in almost all cases (apart from clone 55-5 which has a particular behavior), the resveratrol concentration drops drastically 60 hours after induction .
d) Abiotic stress (UV) study carried out on vitroplants in culture on agar medium
This study, carried out on a plant growing on agar medium, makes it possible to analyze the production of resveratrol under the condition of possible expression of other mechanisms of defense of the plant, at least those likely to be expressed in the culture conditions. in glass. In addition, it allows to follow the resveratrol synthesis over a longer period (in previous studies, the isolated leaf necroses beyond 72 hours).

Four clones of cultured in vitro cultured 41B (55-2, 3, 5, 6) were treated with UV under the same conditions as before (8 min at 254 nm), the leaves being taken from the plant only. 20 hours after induction. The results obtained were compared with the non-transformed control as well as with a clone of
Vitis rupestris and 3 grape clones Vitis vinifera, known in the field to have a sensitivity more or less to attack Botrytis on berries.

 The literature (SBAGHI et al., 1993) shows that a correlation exists between the susceptibility of berries to Botrytis, evaluated at the vineyard, and the resveratrol content of vitroplant leaves induced in the U.V.

The results are shown in Table 6 below.

Table 6: Results of resveratrol assays by
HPLC carried out 20 hours after induction 8 minutes UV 254 mn on different vine varieties.

<Tb>

<SEP> Variety <SEP> tested <SEP> (resveratrol)
<tb><SEP> Ag <SEP> g <SEP> 1 <SEP> weight <SEP> sec
<tb> Rupestris <SEP> 215 <SEP> 351
<tb> Rootstock <SEP> 41B <SEP> 237
<tb> Ugni-white <SEP> 479 <SEP> 209
<tb> Pinot <SEP> black <SEP> 386 <SEP> 86
<tb> Crazy <SEP> white <SEP> 280 <SEP> 37
<tb> Pct <SEP> 55-2 <SEP> 361
<tb> Pct <SEP> 55-3 <SEP> 235
<tb> Pct <SEP> 55-5 <SEP> 115
<tb> Pct <SEP> 55-6 <SEP> 539
<Tb>
Legend of Table 6
The UV induction is carried out on vitroplants in culture on agar medium. The leaves are removed and extracted for analysis 20 hours after treatment.

PCT 55-2, 3, 5, 6 - transformants having incorporated the 13 kb insert into their genome. The results represent the average of 3 repetitions.

These results indicate that a correlation exists between varieties and grape varieties between sensitivity to
Botrytis and resveratrol content of leaves 20 hours after induction. Two groups are identifiable among the unprocessed varieties and grape varieties. The first, consisting of
V. ruspestris, V. vinifera x V. berlandieri (41B) and Ugniblanc, correspond to those who are relatively tolerant to the fungus. The second, made up of Pinot Noir and La Folle Blanche, represents those considered as moderately tolerant or even very sensitive (Crazy white for example).

 The transformants, on the other hand, have, on average, levels of resveratrol, 20 hours after induction, greater than or equal to the non-transformed control 41B (539, 362 and 236 CLg dry matter respectively for clones 55-6, 55-2). and 55-3 and 237 for the witness).

 For the 55-5 transformed clone, however, the concentration obtained is half that of the control. If we compare these values, expressed in Agg 1 of dry weight, with those of the isolated leaves (table 5), one realizes that they are similar for the control but on the other hand that the variations exist for the transformants analyzed 20 hours after induction. These are sometimes very important (135 for single-leaf induction and 362 for vitroplant induction for clone 55-2, 44 against 236 for clone 55-3, 392 against 116 for clone 55-5 and 339). against 540 for 55-6). Moreover, a great variability exists between the different repetitions.

 2) Comparative Study of the Expression of the Vst Gene and Its Biotic Stress Induction Kinetics on Grapevine Vitroplants 41B, Transformed by the 13 kb Insert and by the Construction Comprising Only the vstl Gene Under the Control of the PR10 PMs Promoter -1.

 The above results (chapter 1), obtained on vitro plants having integrated by genetic transformation additional copies of the stilbene synthase genes (in the form of a 13 kb insert comprising two functional sequences vst1 and vst2), show that Overproduction of resveratrol, product of the reaction catalyzed by the enzyme stilbene synthase, is possible in some transformants when these genes are under control of their own promoters and in response to abiotic stress such as UV irradiation.

With respect to these results, the production of resveratrol on two types of transformants was then verified, the first representing a 41B clone having integrated the 13 kb insert (stilbene synthase vst1 and vst2 genes under the control of their own promoters). , the latter several clones having incorporated the vstl gene alone under the control of the PMs-PR10-1 alfalfa defense gene regulatory sequence. This comparison was made after induction by biotic stress caused by
Botrytis cinerea.
a) Methods used
a) Demonstration of the fonsitoxicity of the resveratrol molecule on Botrytis cinerea
The literature data on the molecule as fungitoxic are contradictory. According to DAI (1994), resveratrol shows an inhibitory action on the development of zoospores of Plasmopora viticole (agent Mildew). On the other hand, according to PONT and PEZET (1990), it does not block the dilution of resveratrol from 10-1 M to 3.7 × 10 -3 M and incubated at room temperature for 7 days. The results showed an inhibitory action of the molecule (IC 50 = 500 mol.l1), with exponential decrease in the average growth diameter of the fungus for concentrations greater than 125 CLmoles.ll. On the other hand, concentrations of 37 Amoles.l 1 inhibit only very little the growth of the mycelium (see Figure 9). This inhibition is however gradually lifted after 7 days of culture under these conditions, moreover. very favorable to the growth of the mushroom. After 20 days, the fungus eventually contaminates the entire culture surface
ss) Tolerance Screening Test of Vitroplant Vine Botrytis cinerea
The test developed consisted of inoculating seedling leaves, grown in vitro on microbouturing medium, by depositing on their upper side of 20 μl a suspension of conidia at 1 × 10 -4 conidia.ml -1 (200 conidia per ml). deposition) in a malt-glucose medium. The seedlings, thus inoculated on 4 different leaves, are then grown in a climatic chamber (photoperiod 16 h day, 8h night, temperature 240C, humidity 70 W). Two days after inoculation, the fourth-ranked leaves, the youngest ones, were observed (necrosis and macerations present counted by a camera connected to a television monitor) and extracted with methanol to allow dosing of resveratrol synthesized in response. on the attack by Botrytis. At 5 days, leaves number 2 were taken to be observed by fluorescence microscopy. Macroscopic observation (necrosis and maceration on the leaves) and enumeration of the leaves with fruiting bodies of the fungus (conidiophores) were also carried out on the leaves number 3. Finally, at 9 days, leaves in interaction with the mushroom, taken on three different plants, were extracted by methanol to assay the resveratrol.
y) Induction of biotic stress by deposition of suspension containing Botrytis cinerea spores on viltroplant leaves - Botrytis tolerance test
This direct confrontation test was carried out according to the technique already described (see previous paragraph).

It is the same for the observations made. For each variety or clone transformed, four vitroplants were used and for each of them three developed leaves of rank 2, 3, 4 were inoculated by the suspension of conidia (200 conidia in 20 μl of malt-glucose medium). Twelve inoculations were therefore performed for each variety or clone and the following analyzes and observations were made Two days after inoculation
- observation of rank 4 leaves for leaf symptoms (zone of maceration of the fungus or necrotic spots, consisting of small black-brown areas, located around mushroom spores, or without visible symptoms), and
- dosage of resveratrol on these same leaves.

Five days after inoculation
- observation of rank 3 leaves for leaf symptoms with enumeration of those carrying conidiophores (fruiting bodies of the fungus), and
- observation by fluorescence microscopy of leaves of rank 2 for localization of the resveratrol synthesis zones.

Nine days after inoculation
- Resveratrol dosage in three leaves, from three different plants, interacting with the parasite.

For each experimental series, three grape varieties of
Vitis vinifera with different susceptibilities to Botrytis attack on berries were studied: Crazy white (sensitive), Pinot noir (moderately tolerant),
Ugni-blanc (tolerant) and 41B (tolerant) rootstock and four of its transformants: one having inserted supernumerary copies of genes encoding a stilbene synthase (insert 13 kb), clone 55-3 and all three others, representing transformants of the construction promoter
PMs PR10-l-vst1 gene are clones 145-2, 145-5 and 1456.
b) Results obtained
The results are presented in Tables 7 and 8, for leaf symptoms observed 2 or 5 days after inoculation and, in Tables 9 and 10, for resveratrol dosages reported either on dry weight or on chlorophyll content. .

Table 7: Macroscopic Observations of Vitroplant-Botrytis cinerea leaf interactions at 2 days

<tb><SEP> Number <SEP> of <SEP> leaves <SEP> n "<SEP> 4, <SEP> observed <SEP> on
<tb><SEP> Variety <SEP> tested <SEP> 4 <SEP> different <SEP> plants, <SEP> where <SEP> appear
<tb><SEP> the following <SEP>SEP> symptoms
<tb><SEP> Zones <SEP> of <SEP> Spots <SEP> None
<tb><SEP> maceration <SEP> necrotic <SEP> symptom
<tb><SEP> visible
<tb> Crazy <SEP> white <SEP> 280 <SEP> 4 <SEP> 1 <SEP> 0
<tb> Pinot <SEP> black <SEP> 386
<tb> Ugni-white <SEP> 479 <SEP> 1 <SEP> 2 <SEP> 1
<tb> 41B <SEP> 0 <SEP> 4 <SEP> 0
<tb> Pct <SEP> 55-3 <SEP> 2 <SEP> 3 <SEP> 0
<tb> Pct <SEP> 145-2 <SEP> 0 <SEP> 3 <SEP> 1
<tb> Pct <SEP> 145-5 <SEP> 0 <SEP> 1 <SEP> 3
<tb> Pct <SEP> 145-6 <SEP> 1 <SEP> 1 <SEP> ~ <SEP><SEP> 2
<Tb>
Legend of Table 7
- Maceration zones: wide and diffuse zone where
Botrytis quickly destroys plant cells. These areas have a light brown beige color.

 - Necrotic spots: very small areas circumscribed around the mushroom. These spots are black-brown.

Table 8: Macroscopic Observations of Vitroplant-Botrytis cinerea leaf interactions at 5 days

<tb><SEP> Number <SEP> of <SEP> leaves <SEP> n0 <SEP> 3, <SEP> observed <SEP> on
<tb><SEP> Variety <SEP> tested <SEP> 4 <SEP> different <SEP> plants, <SEP> where <SEP> appear
<tb><SEP> the following <SEP>SEP> symptoms
<tb><SEP> Zones <SEP> of <SEP> Spots <SEP> None
<tb><SEP> maceration <SEP> necrotic <SEP> symptom
<tb><SEP> visible
<tb> Crazy <SEP> white <SEP> 280 <SEP> 4 <SEP> 0 <SEP> 4
<tb> Pinot <SEP> black <SEP> 386 <SEP> 3 <SEP> 1 <SEP> 2,5
<tb> Ugni-white <SEP> 479 <SEP> 2 <SEP> 1 <SEP> 0.5
<tb> 41B <SEP> 1.5 <SEP> 2 <SEP> 0.5
<tb> Pct <SEP> 55-3 <SEP> 2 <SEP> 2 <SEP> 1
<tb> Pct <SEP> 145-2 <SEP> 1.5 <SEP> 2 <SEP> 1
<tb> Pct <SEP> 145-5 <SEP> 0.5 <SEP> 4 <SEP> 0
<tb> Pct <SEP> 145-6 <SEP> 3 <SEP> 2 <SEP> 1
<Tb>
Legend of Table 8
- Maceration zones: wide and diffuse zone where
Botrytis quickly destroys plant cells. These areas have a light brown beige color.

 - Necrotic spots: very small areas circumscribed around the mushroom. These spots are black-brown.

Table 9: Results of resveratrol assays by
HPLC on different vine varieties interacting with Botrytis cinerea for 2 days

<tb><SEP> Variety <SEP> Resver. <SEP> mg.gl <SEP> Resver. <SEP> g.g1 <SEP>
<tb><SEP> tested <SEP> chloroph. <SEP> weight <SEP> sec
<tb> Crazy <SEP> B280 <SEP> 5.1 <SEP> 101
<tb> Pinot <SEP> N386 <SEP> 4.3 <SEP> 102
<tb> Ugni <SEP> B479 <SEP> 3.9 <SEP> 86
<tb> G41B Holder <SEP> 5.7 <SEP> 112
<tb> Pct <SEP> 55-3 <SEP> 4.6 <SEP> 118
<tb> Pct <SEP> 145-2 <SEP> 6.9 <SEP> 170
<tb> Pct <SEP> 145-5 <SEP> 2.2 <SEP> 83
<tb> Pct <SEP> 145-6 <SEP> 4.3 <SEP> 107
<Tb>
Legend of Table 9 - Resver. : resveratrol; chloroPh. : chlorophyll
Mad B 280: White mad 280; Pinot N 386: Pinot noir 386; Ugni B 479: Ugni-white 479; Holder g41 B: Holder 41B.

Table 10: Results of resveratrol assays by
HPLC on different vine varieties interacting with Botrytis cinerea for 9 days

<tb><SEP> Variety <SEP> tested <SEP> resveratrol <SEP> mg.g <SEP> 1 <SEP> resveratrol <SEP> ug.g- <SEP>
<tb><SEP> chlorophyll <SEP> weight <SEP> sec
<tb> Crazy <SEP> White <SEP> 280 <SEP> 4.0 <SEP> 38
<tb> Ugni-white <SEP> 479 <SEP> 31.7 <SEP> 145
<tb> Rootstock <SEP> 41B <SEP> 11.9 <SEP> 59
<tb> Pct <SEP> 145-5 <SEP> 602.1 <SEP> 2558
<Tb>
Two days after inoculation
The observation focused on the youngest leaves (rank 4). In almost all cases, except for 41B and clones 145-2 and 145-5, maceration zones were observed (colonization of the fungus). The susceptible varieties have presented more than the tolerant grape varieties
Crazy white and Pinot noir respectively 4 and 3 zones against 1 only for the grape Ugni-white. Only transformants 145 (PMs promoter PR10-1-vstl gene) and Ugni-blanc gave leaves without visible symptoms. The necrotic zones, defensive reaction of the plant, appeared mainly on 41B and its transformants and, for the grape varieties, on Pinot noir and Ugniblanc.

 When comparing these observations to resveratrol assays (Table 9), no correlation can be established since, based on dry weight, the resveratrol contents of these leaves are comparable with about 100 g / kg dry matter. They are established for all the vitroplants examined at values between 4 and 5 mg.g-l chlorophyll.

 Only the rootstock 41B and above all the transformant 145-2 have higher values. Clone 145-5 has a lower value (about half).

 A comparison can also be made with resveratrol levels obtained previously 20 hours after induction of vitroplants with U.V. (Table 6). Although the sampling period is not comparable (20 hours for abiotic stress against 48 hours for biotic stress, but the time required for spore germination must be taken into account), lower levels of resveratrol after a biotic stress (half or a third, the case of Ugniblanc), except for the Pinot Noir where it is comparable and for the Mad White where it is about three times stronger.

In all the samples analyzed, it is possible to emphasize a strong dispersion of the values obtained in the different repetitions made. This variability between different leaves of the same plant, already observed with the samples having undergone UV inductions, could, again, have several origins. Among the most plausible hypotheses, one can quote
A great variability of reaction between vitroplants of the same clone against aggression of the fungus. The variable leaf symptoms, obtained after inoculation with Botrytis spores, seem to confirm this (variability in infection, in the physiological state of each plant, etc.).

 The assay, carried out on the whole leaf, is not representative of the variations of synthesis of resveratrol existing at the level of each cell that composes it. It is generally accepted that in hypersensitivity reactions to a parasite, not all limb cells synthesize defense molecules. Only cells located near the attack zones of the fungus are induced to the synthesis of phytoalexins. The analyzes carried out in this test therefore represent only values corresponding to an average level of resveratrol present in the leaf limb in interaction with the parasite. A significant dilution phenomenon can therefore occur, especially for resistant plants, between the concentrations present in the cells induced in the synthesis of phytoalexin and the value found during the analysis of the whole leaf. This is probably more marked in the first period of expression of the defense reactions of the plant.

Five days after inoculation
The observations of the symptoms show (photo sheets 1 and 2 (Figures 10 and 11) and table 8) that maceration zones are formed, in greater or lesser numbers, in all the grape varieties, rootstocks and transformants studied. These are often associated with the presence of conidiophores (fruiting bodies of the fungus). Among the grape varieties and the 41B control rootstock, La Folle Blanche, the most sensitive species to
Botrytis, has attacks on all the studied leaves and also conidiophores. If we establish a hierarchy in the gravity of the symptoms then come the Pinot noir, then the Ugni-blanc and finally the 4113.

However, for these last two, conidiophores are developed on only half a leaf. As for the transforming clones, three clones gave more or less similar reactions: 55-3, 145-2 and 145-6. Only clone 145-5 showed a good tolerance to the parasite, since only half a leaf allowed the development of a maceration zone and no conidiophore is visible.

 On the other hand, for this clone, the majority of the leaves reacted to the aggression by forming necrotic zones. An example is also presented on the photo board 2 (Figure 11).

To verify whether these results corresponded to significant differences in the induction of resveratrol synthesis and thus to the expression of gene (s) encoding a stylbene synthase, leaves, inoculated with Botrytis spores, were observed. fluorescence microscopy (equipped with filter A: excitation filter from 340 to 380 nm, stop filter at 425 nm). Under these conditions, chlorophyll fluoresce red, resveratrol bluish white or darker blue depending on its concentration. Two grape varieties, Folle blanche (sensible) and
Pinot noir (moderately tolerant), rootstock 41B (tolerant) and one of its transformants 145-5 (construction
Promoter PMs PR10-1-vstl gene) were studied by this method. The photographs of these observations are presented on Photo Plate 3 (Figure 12). Remarkable differences are visible. On the white mad, the mycelium of the parasite (black in the photo) has developed and colonized the tissues. Virtually no bluish cell is observable. On the Pinot Noir grape variety there is a bluish zone forming a barrier around and in the zone of inoculation and beginning of colonization of the mushroom. The growth of the mycelium is slowed down or even blocked by this barrier, even if a process of maceration of the tissues has already been initiated. A more intense blue color also exists in the veins. On the control rootstock 41B control, in the studied area, scattered cells, distributed throughout the limb, show a clear blue fluorescence with a greater intensity in the veins. No colonization process of the fungus has been initiated and scattered necrotic zones, limited to a few cells, are visible. On the 41B transformed by PMs promoter PR10-1-vstl gene, the results are spectacular for the 145-5 clone. An early colonization of the tissues by the fungus was initiated (black zone) but, very quickly, a cellular barrier, synthesizing resveratrol, was formed around this zone, thus blocking its extension. In the maceration zone of the fungus blue cells are still visible. In addition, a large part of leaf limb cells that are not in contact with the fungus also synthesized resveratrol.

The veins also have an intense blue color.

 These observations demonstrate that there is therefore a correlation between the intensity of leaf symptoms, developed after inoculation of vitroplants by Botrytis spores, and the stilbene-type phytoalexin content of the leaves. The most tolerant plants are those with a resveratrol synthesis distributed over a large part of the blade. This is done with the 145-5 transformant, having a gene encoding a stilbene synthase under the control of the PR10-1 PMs promoter, isolated from alfalfa.

Nine days after inoculation
Observations of the symptoms showed that Folle blanche vitroplants were also particularly sensitive to Botrytis attacks in this test. At nine days, they are all overgrown with the fungus and in most cases have a severe degradation of their chlorophyll. As regards the Ugni-blanc grape variety and the 41B rootstock, no difference could be found in the expression of leaf symptoms. In general, the results of the observations of the symptoms carried out at five days are found in a little more developed for the zones of maceration. On the other hand, leaves with conidiophores became necrotic in most cases.

Four varieties were analyzed for their resveratrol content: the white mad (susceptible), Ugni-blanc (tolerant), the 41B (tolerant) and the 145-5 (41B transformed).
Promoter PMs PR10-l-gene vstl). For the 145-5, nine days after inoculation, he still did not show any noticeable symptoms of Botrytis attack. The results of the resveratrol content assays are shown in Table 10. Expressed in pg of resveratrol.g.l of dry weight or in mg.g of chlorophyll, resveratrol concentrations allow the following classification of varieties, ranging from the lowest value towards the strongest
White mad <41 B Ugni-white <41B transformed 145-5.

 The differences in concentration are very important, since the 145-5 transformant has a value nearly 43 times greater than that of the control 41B (in c -1 dry weight) and 50 times higher if it is expressed in gl of chlorophyll . The assays thus confirm the evaluations carried out by fluorescence microscopy at five days (see photo plate 3, FIG. 12).

With regard to Ugni-blanc and 41B, Table 10 shows that the former has approximately 3 times more resveratrol than the latter, whatever the units chosen. However, these two varieties react identically in terms of leaf symptoms. Other phenomena than the synthesis of resveratrol can be put into play to explain this similarity of tolerance. It should also be noted that under the conditions of the test, the plant-Botrytis confrontation is particularly favorable to the latter. The environmental conditions existing in an in vitro container make that after about 20 days of culture, all the vitroplants whatever they are, are infected by the fungus.
c) Conclusions on the genetic transformation of visna and the analysis of the efficacy of the promoters studied
The experiments on 41B embryogenic cells (V. Vinifera x V. Berlandieri hybrid rootstock) transformed with Agrobacterium tumefaciens comprising the different constructs, did not make it possible to regenerate plants with the constructs which included the promoter-controlled vst1 gene. 35S or its derivatives.

No strong constitutive activity on the whole plant could thus be obtained.

 On the other hand, vine plants having incorporated the PMs PR10-1-vst1 gene and 13 kb insert constructs (vstl and vst2 genes under the control of their own promoters) were obtained. These plants, transformed genetically, could be compared to the control (41B unconverted) and multiplied by micropropagation.

 Compared to untransformed controls, few differences could be observed under UV stress (biotic stress) in the resveratrol concentrations of the various transformants obtained with the 13 kb insert, where are present in the sequence used as insert two vstl genes and vst2, genes both under control of their own promoters.

 On the other hand, the results show that the chimeric PMs PR10-l-vst1 gene allows overexpression of the phytoalexin resveratrol (product of the reaction catalyzed by a stilbene synthase), in the presence of a biotic stress caused by Botrytis cinerea of the order of 50 times more, 9 days after infection. These plants then show better tolerance if they are compared either to the control or to transformants obtained with the 13 kb insert.

 Thus, the transformation with the construction associating the alien PM4 PR10-1 inducible promoter with the stilbene synthase genes makes it possible to overexpress the stilbene synthase genes in grapevine plants in response to a stress, for example the attack of a pathogen.

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Claims (29)

 A nucleic acid comprising the promoter sequence of a PR alfalfa protein associated with at least one sequence of a gene encoding a stilbene synthase.  2. Nucleic acid according to claim 1, characterized in that the promoter of a PR protein alfalfa is an inducible promoter in plants, specific tissues or not, by biotic or abiotic stress.  3. Nucleic acid according to one of claims 1 and 2, characterized in that the promoter sequence of a PR alfalfa protein is selected from the group consisting of a) IND S1 sequence, b) any sequence corresponding to a fragment of IND S1 sequence and having promoter sequence effect in plants.  4. Nucleic acid according to claim 3, characterized in that the promoter sequence of a PR protein alfalfa has at least 80 W of homology with the sequence IND S1.  5. Nucleic acid according to claim 3, characterized in that the promoter sequence of a PR protein alfalfa has at least 90 k of homology with the sequence IND S1.  6. Nucleic acid according to claim 3, characterized in that the promoter sequence of a PR protein alfalfa has at least 95 k of homology with the S1 IND sequence.  7. Nucleic acid according to one of claims 1 to 6, characterized in that the gene sequence encoding a stilbene synthase is selected from genes isolated from the genomes of peanut, orchid, grapevine. and pine.  8. Nucleic acid according to claim 7, characterized in that the gene sequence encoding a stilbene synthase is the sequence of a gene encoding a vine stilbene synthase.  Nucleic acid according to claim 8, characterized in that the sequence of the gene coding for a vine stilbene synthase is a sequence chosen from  a) the vstl gene,  b) the vst2 gene.  10. System for expressing a stilbene synthase gene in plants, characterized in that it comprises at least one nucleic acid according to one of claims 1 to 9.  11. System for expressing a stilbene synthase gene in plants according to claim 10, characterized in that it is a vector.  12. Expression vector according to claim 11, characterized in that it is a plasmid.  13. Expression system according to one of claims 10 to 12, characterized in that it can be transferred into Agrobacterium strains.  14. Expression system according to one of claims 10 to 13, characterized in that it is inducible in plants by biotic or abiotic stress.  15. Expression system according to claim 14, characterized in that the biotic stress is the attack of a parasite.  16. Expression system according to claim 15, characterized in that the parasite is a bacterium, a yeast, a fungus or a virus.  17. Expression system according to claim 16, characterized in that the parasite is Botrytis cinerea or Plasmopora vi ticola.  18. Expression system according to claim 14, characterized in that the abiotic stress is a mechanical injury.  An expression system according to claim 18, characterized in that the mechanical wound is caused by an insect.  20. Expression system according to claim 18, characterized in that the mechanical injury is caused by a physical phenomenon such as wind or frost.  21. Plant cell transformed by a system or a vector according to one of claims 10 to 20.  22. Cell according to claim 21, characterized in that it is a vine cell.  23. A method for obtaining a cell according to one of claims 21 and 22, characterized in that transforms a plant cell using a microbiological process including a system or vector according to one of claims 10 to 20.  24. Process for obtaining plants expressing a stilbene synthase gene, characterized in that plant cells of said plants are transformed using a system or a vector according to one of claims 10 to 20, cells expressing said gene are selected and a plant is regenerated from these cells.  Plant comprising an expression system according to one of claims 10 to 20.  Plant comprising cells according to one of claims 21 and 22.  Plant obtained by the implementation of a method according to one of claims 23 and 24.  28. Plant according to one of claims 25 to 27, characterized in that it is a plant of agronomic interest.  29. Plant according to claim 28, characterized in that it is about the vine.